1. Field of the Invention
The present invention relates to a method for the crystallization of proteins using nanoparticles as nucleation agents. Particularly, the invention relates to a method to increase the opportunity of crystallization of protein by inducing nucleation of protein particularly by conjugating His-tag to protein in order for the protein to be combined with nanoparticles having carboxyl group using Ni2+ ions as a linker.
2. Description of the Related Art
X-ray crystallography is a method to provide an important clue for the understanding of functions of biopolymers including proteins through structure determination in the field of structural biology. The structures of 89% of the total biopolymers identified so far were determined by X-ray crystallography. Structure determination using X-ray crystallography is accomplished by the following steps such as protein over-expression, purification, crystallization, collection of X-ray diffraction data, and structure determination. Supersaturation of protein, which is necessary for protein crystallization, can be induced by increasing the concentration of protein or by increasing a reagent to induce protein precipitation (FIG. 1). However, the concentrations of protein and the precipitation solution have to be precise and suitable for inducing nucleation. If the concentration is too high, an amorphous precipitate would be generated. On the other hand, if the concentration is too low, the solution would stay in metastable state so that a precipitate would not be generated at all. Spontaneous protein crystallization is composed of two steps, which are nucleation and crystal growth (FIG. 2). For the formation of stable nucleation, proteins existing as a monomer in the solution have to be gathered together to form a cluster. Once nucleation occurs, crystal growth continues as long as the protein stays in the supersaturation state. Factors affecting the crystallization of protein such as precipitants, pH, temperature, pressure, and gravity, have been studied. However, the conditions for the crystallization of each protein vary (Adachi, H. et al., Temperature-screening system for determining protein crystallization conditions. Jpn. J. Appl. Phys. 44, 4080-4083, 2005), and many trials and errors to find out proper crystallization conditions for each protein are still needed. In the course of crystallization, nucleation is the most important step. So, inducing nucleation can be an important way to increase the chance of successful crystallization of protein.
Significant advances have been made in many stages of X-ray crystallography due to the technological developments of modern biology. However, success in crystallization of protein is still depends on the time consuming trial-and-error process.
Therefore, the present invention provides a method for using nanoparticles in order to help nucleation that plays an important role in protein crystallization. In the previous studies, induction of nucleation using gold nanoparticles has been reported. However, the method is a non-selective method using the conjugation between gold particles and sulfur atoms included in cysteine or methionine residues exposed on the surface of protein. So, this method can only be applied to those proteins that have sulfur exposed on the surface. This method also has a problem of non-selective conjugation between gold particles with other impurity proteins in addition to the target proteins. Thus, it was difficult to induce nucleation of target protein with high purity that could be a great help for the crystallization of protein. The present inventors confirmed that the method for inducing nucleation using gold nanoparticles could be effectively used as a method for the crystallization of protein with high purity, leading to the completion of the invention.